The highly endangered European pond turtle (Emys orbicularis) was reintroduced in Switzerland in 2010. Up until 2019, no routine medical examinations have been carried out prior to its release or during recapture events. The aim of this study was to assess the health status of captive and free-living Emys orbicularis populations in Switzerland, taking into account the most important and frequently occurring health threats to freshwater turtles. A total of 141 European pond turtles, including captive (n = 89) and free-living (n = 52) individuals, underwent clinical examination (n = 136), choanal and cloacal swab collection for microbiology investigation (n = 140), blood sampling (n = 121), fecal examination for parasitology (n = 92), radiography (n = 84), and ultrasound (n = 46). Microbiology investigation included conventional PCR for herpesvirus, ranavirus, and Mycoplasma spp. Blood was used for the establishment of reference values for hematocrit, leukocyte count, and differential blood count as well as for biochemistry parameters tested with the VetScan VS2. An emydid Mycoplasma was detected in 40% (n = 56/140; 95%CI: 31.82-48.61%) of the turtles, including one individual with upper respiratory signs. Four animals positive for Mycoplasma arrived dead or were euthanized during the study period. Their necropsies revealed no evidence of respiratory disease. No ranavirus or herpesvirus was detected in any of the tested turtles. Two presumptively fatal infections with spirorchiid trematodes were reported during the study period. Endoparasites were detected in only 7.94% of the samples examined. This study provides comprehensive data on the current health status of the largest sample size of captive and free-living populations of Emys orbicularis ever assessed to date and serves as a baseline for future research investigations and management recommendations in this species.